Experimental Study Of Distribution, Phenotype,Differentiation, Recruitment, And Clinical Significance Of CD4+â…¡-9+T Cells In The Tumor Microenvironment Of Patients With Colorectal Cancer

Part1Distribution and clinical significance of CD4+IL-9+lymphocyte in immunol micro-environment of colorectal cancer and peripheral bloodObjective:to detect the distribution of CD4+IL-9+(Th9) lymphocyte in immunol microenvironment of colorectal cancer (CRC) and analyze its relation with clinical pathology and other CD4+T subtype.Methods:Firstly, carcinoma tissue and relevant adjacen normal tissue (ANT) of5patients with CRC were enrolled for identification of protein expression of CD4and IL-9by Immunofluorescence technique. Other45patients with CRC who underwent operation for the first time were enrolled, carcinoma or ANT, pre or post-operative peripheral blood (PB) of these patients, and PB from15healthy volunteers was detected for the proportion of CD4+IL-9+T, Thl, Th2, Th17in CD4+T lymphocyte through flow cytometry (FCM); Clinic pathologic data like sexy, age, nation, Duke’s stage, tumor size, tumor location and carcinoma metastasis of lymphocyte, lever of CEA and AFP was recorded. The proportion of CD4+IL-9+T in tumor, ANT, pre or post-operative, healthy blood was compared. Correlation of distribution between CD4+IL-9+T and Th1, Th2, Th17was analyzed.Results:Immune double staining indicated that CD4+IL-9+T existed in the tumor and ANT of patients with CRC; proportion of CD4+IL-9+T in carcinoma tissue was significantly higher than that in ANT and pre or post-operation and healthy PB(1.12±0.33%,0.85±0.29%,0.65±0.27%,0.43±0.25%, respectively), P<0.05; proportion of CD4+IL-9+T in pre-operational PB was higher than that of post-operation,p<0.05; CD4+IL-9+T lymphocyte is correlated with Duke’s stage, p<0.05, while there was no significant relation between its expression and other factors; there was not significant relation between the proportion of CD4+IL-9+T in tumor tissue and Thl, r=-0.140,p=360, while negative correlation existed between CD4+IL-9+T and Th2, positive correlation existed between CD4+IL-9+T and Th17, r=0.367,p=0.013.Conclusion:CD4+IL-9+T existed among tumor environment of CRC, and there is enrichment phenomenon of CD4+IL-9+T in the immune microenvironment of CRC, CD4+IL-9+T is correlated to the progression of CRC and may take part in regulating the immune microenvironment of CRC and act as a key role in the progress of CRC. Part2The phenotype, relevant cytokine expression and clinical significance of CD4+IL-9+cells in tumor microenvironment of CRCObjective:To explore phenotype, expression of relevant cytokine, transcription factor and chemokine of CD4+IL-9+T in the tumor environment of patients with CRC, and discuss the related clinical significance.Methods:45patients mentioned above were enrolled, samples from carcinoma and related adjacent nomal tissue (ANT), pre or post-operative peripheral blood (PB) of these patients, and PB from15healthy volunteers were gathered. RT-PCR technique was applied to detected the expression of cytokine IL-9and IL-10, transcript factor PU.1and IRF4, chemokine CCL5, CCL2, CCL22, CCL20,CCL27in the samples above; Liquid protein chip technology was applied to detect concentration of cytokine IL-9and IL-10in the sample above; tumor tissue from another10patients with CRC in the same term confirmed by pathology were selected to undergo FCM for determining expression of phenotype HLA-DR, CD25, PD-1, Foxp3, CD45RO, CD45RA and chemokine receptor CCR1, CCR2, CCR3, CCR4, CCR5, CCR6.Results:CD4+IL-9+T cell in the tumor environment of CRC showed low expression of phenotype HLA-DR(5.4±2.9%), CD25(1.7±1.3%), PD-1(4.9±3.2%), Foxp3(2.5±1.6%), CD45RA(2.3±1.8%), but high expression of CD45RO(83.2±31.5%); Low expression of chemokine receptor of CCR1(7.89±3.42%), CCR3(3.15±1.56%), CCR4(8.69±4.25%), CCR6(2.56±1.34%), while high expression of CCR2(79.53±24.12%), CCR5(72.34±21.69%). The results of RT-PCR indicated that the relative expression of IL-9in tumor tissue (2.21±1.634) was higher than that of ANT (1.38±1.68), and also higher than in pre-or post-operational and healthy PB,P<0.05; Expression of IL-9of pre-operational PB was higher that of post-operational and healthy PB, P<0.05. Similar results existed when expression of IL-9were compared among different samples.The results of liquid protein chip showed that concentration of IL-9in the tumor micro-environment (0.82±0.65pg/ml) was higher than that in ANT (0.59±0.53pg/ml) and pre-or post-operational and healthy PB,[<0.05; Similar results came out when concentration of IL-10were compared among different samples. Correlation test showed that protein level of IL-9was correlated with Duke’s stage, P<0.05, while there was not significant relation between it and other clinical-pathological factor like age, sex, nation, lymphatic metastasis, carcinoma location, carcinoa size and degree of differentiation,P>0.05; there was not significant correlation between protein level of IL-10and clinical pathology.RT-PCR indicated that expression of chemokine CCL5in the tumor was higher than that in ANT and pre-or post-operational or healthy PB,P<0.05; the expression of CCL22in carcinoma tissue was lower than than in ANT,P<0.05, while the expression of CCL2in tumor tissue was higher than that in pre-or post-operational or healthy PB,P<0.05; it showed same results with CCL2in regard of chemokine CCL20, CCL22, CCL27. The results of RT-PCR indicated expression of PU.1in the tumor tissue (1.12±0.30) was higher than that in ANT (0.86±0.42),P=0.005; There was no significance of expression of IRF4between tumor tissue (0.45±0.11) and ANT (0.43±0.10),P=0.392. Pearson correlation analysis showed there was positive correlation among expression of PU.1and CD4+IL-9+T (r=0.346,P=0.020) while no significant correlation existed between expression of IRF4and CD4+IL-9+T.Conclusions:CD4+IL-9+T in the micro-environment of CRC has its own phenotype including the T cell function markers and chemokine receptor expression profiles. Relative cytokine IL-9and IL-10, chemokine RANTENS, transcript factor PU.1, chemokine receptor CCR2and CCR5of CD4+IL-9+T was highly expressed in the tumor micro-environment. Interaction of RANTENS and CCR5may be one of the mechanisms of CD4+IL-9+T recruitment in the tumor micro-enviroment.Part3The mechanism of differentiation and recruitment of CD4+IL-9+T cells in tumor microenvironment of CRC and the role of IL-9and IL-10on proliferation, apoptosis and migration of SW480cell lineObjective: to analyze the mechanism of differentiation and recruitment of CD4+IL-9+T cells in tumor microenvironment of CRC and the role of IL-9and IL-10on proliferation, apoptosis, migration and invasion about SW480cell line. Methods:we used magnetic activated cell sorting to separate naive CD4+T cells from tumor micro-environment of CRC, then IL-4and TGF-β was added alone or both for incubation. FCM and Real time-PCR were respectively applied to determine proportion of CD4+IL-9+T, expression of cytokine IL-9and IL-10, transcript factor PU.1and IRF4in different groups, difference between different groups was compared. Chemotaxis test was conducted to analyzed the recruitment ability of CCL2, CCL5, CCL22, CCL20for CD4+IL-9T cell from tumor micro environment; cytokine IL-9and IL-10was added along into SW480cell line, we did migration and invasion experiment, proliferation or apoptosis trials to explore the role of IL-9and IL-10on migration, invasion, proliferation and apoptosis of SW480cell.Results:we successfully separated naive CD4+T cell from tumor microenvironment of CRC with high purity (97.7%), the ability that inducing CD4+IL-9+T differentiation of IL-4combined with TGF-β was stronger than IL-4or TGF-β used alone or blank, with CD4+IL-9+T proportion1.35±0.45%,1.72±0.77%,3.47±1.75%,10.7±2.94%after provoked, respectively. Proportion of CD4+IL-9+T in IL-4group was similar with that in blank group, while proportion of CD4+IL-9+T in TGF-β group was higher than that in IL-4group and blank group, P<0.05.RT-PCR indicated that expression of IL-9, IL-10, PU.1, IRF4in mixed group was higher than in blank group, IL-4group and TGF-β group, P<0.05; Expression of IL-9in TGF-β group was higher than blank group,P<0.05, other comparison between group indicated no significant difference. Chemotaxis test indicated that chemokine CCL5、CCL22showed better recruitment effect for CD4+IL-9+T cells than CCL2, CCL20and blank,p<0.05, while the recruitment effect for CD4+IL-9+T cells of CCL2, CCL20was similar with blank.Transwell migration and invasion experiment indicated IL-9could promote migration or invasion of SW480cell lines, and its effect was stronger than IL-10; IL-10showed litter effect on migration and invasion of SW480cell line.FCM showed than both IL-9and IL-10could promote proliferation of SW480cells, proliferation rate of SW480cell in this to group was both higher than that in blank group,p<0.05; apoptosis rate of SW480cell in the IL-9is lower than that in the IL-10and blank group,p<0.05; IL-9showed litter effect on inhibition of apoptosis of colorectal cancer SW480cell.Conclusion:It was confirmed further by our study that TGF-β combined with IL-4could promote naive CD4+T cell differentiating into CD4+IL-9+T and its function was better than be used alone, cytokine can promote migration and invasion about SW480cells, blocking the signal pathway or inhibiting the activity of IL-9may be the new therapeutic target for CRC.